抗汉坦病毒核蛋白单链抗体的构建与表达  

Construction and Expression of Anti-Hantavirus N Protein Single Chain Fv Antibody Fragment in Escherichia coli

作　　者：李建东 王涛 李川 纪燕 梁米芳 

机构地区：[1]中国疾病预防与控制中心病毒病预防控制所,北京100052

出　　处：《病毒学报》2003年第2期118-122,共5页Chinese Journal of Virology

基　　金：国家自然科学基金资助项目(C03011402)

摘　　要：汉坦病毒核蛋白在病毒各个基因组片段核糖核蛋白(RNP)复合物的形成,以及RNP复合物装配入病毒颗粒中发挥重要作用。体外研究表明,核蛋白与病毒RNA的特异性结合结构域位于第175～217个氨基酸残基,羧基端其它部分为非特异性结合结构域。为了在细胞内病毒复制的正常过程中研究核蛋白的功能,应用噬菌体表面呈现技术,分别从鼠杂交瘤细胞和人外周血淋巴细胞天然抗体库中,筛选出两株不同抗原结合位点的抗汉坦病毒核蛋白抗体L13F3和H34Fab抗体,并在大肠杆菌中进行表达。L13F3Fab抗体的抗原结合位点位于核蛋白氨基端25～65个氨基酸之间,H34的抗原结合位点位于核蛋白的羧基端的一半。分别使重链可变区羧基端与轻链可变区氨基端通过9个氨基酸寡肽连接起来,构建成单链抗体,并在大肠杆菌内进行表达。通过酶联免疫吸附试验(EL SIA)、间接免疫荧光试验(IFA)和Westernblot检测,确定所构建单链抗体与母抗体的抗原结合特性无差别。单链抗体分子量小,易于操作并保留了全部的抗原结合活性,是在细胞内探索汉坦病毒核蛋白功能的良好工具。The nucleocapsid (N) protein of Hantaviruses plays an important role in the formation of RNP complex of each genomic segment of vRNA and the assembly of the RNPs into virionPrevious studies showed that the RNA binding domain (RBD) of N protein specifically interacts with vRNA located at amino acids 175 to 217,nonspecific RBD was localized at the carboxyl-terminal amino acids of the N proteinTo elucidate the function of N protein in vivo, we developed single chain Fv antibodies (scFv) with different binding sites at N protein. By using phage display technology,we isolated the heavy and light chain genes of both group specific(L13F3) and Hantann virus(HTNV) type specific(H34) antibodies to HTN virus N protein from mouse hybridoma cell lines and human peripheral lymphocytes respectively,and expressed them in form of Fab in Escherichia coliThe epitope on the N protein recognized by L13F3 located at aa 25 to aa 65 of the N-terminus of N protein,the binding site of HTNV specific antibody H34 may locate at the half of C-terminus of N proteinTo construct scFv,the carboxyl terminus of heavy chain variable fragment was connected with the amino terminus of light chain variable fragment by 9 amino acid flexible linkerThe constructed scFvs were expressed,and analyzed by ELSIA,IFA and Western-blot,the results showed that the scFvs retained their parental antigen binding affinity and specificityThe scFv antibodies to Hantavirus are easy to be operated and further engineered,therefore it would be expected to be a good tool to investigate the function of N protein

关 键 词：汉坦病毒 核蛋白 单链抗体 构建 表达 

分 类 号：R373.32[医药卫生—病原生物学]